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. 2021 Nov 15;24(12):103435.
doi: 10.1016/j.isci.2021.103435. eCollection 2021 Dec 17.

Plant protein reduces serum cholesterol levels in hypercholesterolemia hamsters by modulating the compositions of gut microbiota and metabolites

Li-Tao Tong  1 , Tianzhen Xiao  1 , Lili Wang  1 , Cong Lu  1 , Liya Liu  1 , Xianrong Zhou  1 , Aixia Wang  1 , Wanyu Qin  1 , Fengzhong Wang  1
Affiliations

Plant protein reduces serum cholesterol levels in hypercholesterolemia hamsters by modulating the compositions of gut microbiota and metabolites

Li-Tao Tong et al. iScience. .

Abstract

Plant proteins exert effects of reducing cardio-cerebrovascular disease-related mortality partly via cholesterol-lowering, which was associated with gut microbiota. Here, we verify that there are significant differences in cholesterol levels among hamsters consuming different proteins. The decisive roles of gut microbiota in regulating host cholesterol are illustrated by the fact that the difference in serum cholesterol levels between hamsters feeding with pea protein and pork protein disappeared when treated with antibiotics. The results of cross-over intervention of pea and pork protein show that serum cholesterol levels are reversed with dietary exchange. The corresponding changes in microbiota suggest that Muribaculaceae are responsible for the inhibitory effect of pea protein on serum cholesterol level, whereas the opposite effect of pork protein is due to Erysipelotrichaceae. Moreover, pea protein supplement alters cecal metabolites including arginine/histidine pathway, primary bile acid biosynthesis, short-chain fatty acids, and other lipid-like molecules involved in cholesterol metabolism.

Keywords: Biological science; Endocrinology; Microbiology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Effects of diverse proteins on serum and liver lipid profiles (A–D) Serum levels of TC: total cholesterol (A); LDL-C: low-density lipoprotein cholesterol (B); HDL-C: high-density lipoprotein cholesterol (C); TG: triglycerides (D) (E) Arteriosclerosis index, which was calculated by TC content minus HDL-C content and divided by HDL-C content. (F–I) Liver levels of TC (F); FC: free cholesterol (G); CE: cholesterol ester (H); TG (I). Statistical significance was calculated by Student's t test (∗p < 0.05; ns, not significant).
Figure 2
Figure 2
Changes of gut microbiota composition in response to diverse dietary proteins (A) The composition of gut microbiota in fecal samples from hamsters after 30 days’ administration of diverse proteins at phylum level. (B) The ratio of Bacteroidota and Firmicutes in different groups. (C) The composition of gut microbiota in fecal samples from hamsters after 30 days’ administration of diverse proteins at genus level; taxa with abundance below 1% were presented as others. (D) Gut microbiota significantly different between pea and pork protein groups at phylum and family levels. The bar plots show the abundance of diverse bacteria. Positive differences in mean of relative abundance indicate bacteria with higher abundance in the pea protein group, whereas negative differences indicate bacteria with higher abundance in pork protein group. Statistical analysis was performed by the Wilcoxon test coupling with false discovery rate (FDR) correction. (E–L) Discrepant bacteria at family level in control (E), rice (F), oat (G), soybean (H), pea (I), chicken (J), pork (K), and beef (L). Statistical significance was calculated by Student's t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant). All error bars indicate SE.
Figure 3
Figure 3
Discrepant bacteria and correlation analysis (A) Taxa with linear discriminant analysis (LDA) score 3 or greater from phylum to genus levels in gut microbiota communities of diverse protein groups. (B) Taxa with linear discriminant analysis effect size (LEfSe) representing bacteria from genus to phylum level in different groups. (C) The interconnection networks of gut microbiota at family level in different groups. Species node sizes represent relative abundance at family levels, and edges represent the association patterns of individual family with the dietary protein types. (D) Contents of short-chain fatty acids (SCFAs) in different groups. (E) Redundancy analysis (RDA) of samples from different groups based on OTU data and gut microbial metabolites. (F) Serum lipid profile. (G) Correction analysis of top 20 genera and amino acid composition of diverse proteins. (H) Lipid profiles. (I) Gut microbial metabolites. Statistical significance was calculated by Student's t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant). All error bars indicate SE.
Figure 4
Figure 4
Effects of pea and pork protein with antibiotics treatment on gut microbiota composition and structure (A) The overview of the antibiotics study design. (B–E) Boxplot of alpha diversity indices at OTU level expressed as Shannon index (B), Simpson index (C), Chao index (D), and Ace index (E). (F) Hierarchial clustering of gut bacteria at OTU level. (G) Principal-component analysis (PCA). (H) Principal co-ordinates analysis (PCoA) based on unweighted UniFrac of all groups at 0 and 30 days. (I) Mean proportions of discrepant species at phylum level in different groups by two-group comparisons using Welch’s t test: Pea_Abx_0d versus Pea_Abx_30d; Pork_Abx_0d versus Pork_Abx_30d. (J) Abundance of the discrepant gut microbiota at family level. (K) Serum cholesterol content of hamsters treated with Abx. (L) Liver contents of TC, FC, CE, TG, HMG-CoA; CYP7A1, LDLR, and LPL. Statistical significance was calculated by Student's t test (∗p < 0.05, ∗∗p < 0.01 ∗∗∗p < 0.001). All error bars indicate SE.
Figure 5
Figure 5
Effects of pea and pork protein supplement on cholesterol level in hamsters (A) The overview of the cross-over intervention of pea and pork protein study design. (B–E) Serum levels of TC (B); LDL-C (C); HDL-C (D); and TG (E). (F) Liver concentrations of TC, FC, CE, and TG. (G) Liver concentrations of HMG-CoA, CYP7A1, LDLR, and LPL. Statistical significance was calculated by Student's t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant). All error bars indicate SE.
Figure 6
Figure 6
Changes of gut microbiota composition in response to pea protein and pork protein (A and B) The composition of gut microbiota at phylum level (A) or genus level (B) in different groups; taxa with abundance below 1% were presented as others. (C) Mean proportions of discrepant species on phylum level in different groups at 30 days by two-group comparisons using Welch’s t test: Pea versus Pork at 30 days. (D) Taxa with LDA score 3 or greater from phylum to genus levels in gut microbiota communities of pea and pork protein groups at 30 days. (E) Proportions of gut microbiota on family level with significant difference among different groups at 60 days. Kruskal-Wallis H test coupling with FDR correction was applied to evaluate the significant difference among the four groups at 60 days. (F) Taxa with LDA score 3 or greater from phylum to genus levels in gut microbiota communities of different groups at 60 days. ∗∗p < 0.01, ∗∗∗p < 0.001. All error bars indicate SE.
Figure 7
Figure 7
▪▪▪ Mean proportions of discrepant species at phylum level in different groups at 60 days by two-group comparisons using Welch’s t test: (A–F) Pea versus Pork (A); Pea_Pork versus Pork_Pea (B); Pea versus Pea_ Pork (C); Pork_Pea versus Pork (D); Pea versus Pork_Pea (E); Pea_Pork versus Pork (F). ∗p < 0.05, ∗∗p < 0.01 ∗∗∗p < 0.001. All error bars indicate SE.
Figure 8
Figure 8
Overview of metabolic signatures among all groups (A) Cecal SCFA contents of hamsters with cross-over intervention of pea and pork. (B) PCA of metabolic features among all groups. (C) Numbers of discrepant metabolites in comparison of the two groups. (D) Pie chart based on counts of HMDB chemical taxonomy (superclass) for all metabolites detected in this study class. (E) KEGG pathway classification of metabolites detected and annotated. Statistical significance was calculated by Student's t test (∗p < 0.05, ∗∗p < 0.01; ns, not significant). All error bars indicate SE.
Figure 9
Figure 9
Cecal metabolome alterations (A–C) Comparison between pork and pea groups by heatmap of discrepant metabolites (A); volcano plot of discrepant metabolites (B); discrepant metabolic signatures ranked by variable importance in projection (VIP) score (C). (D–F) Comparision between Pea_Pork and pea groups by heatmap of discrepant metabolites (D); volcano plot of discrepant metabolites (E); discrepant metabolic signatures ranked by VIP score (F). (G–I) Comparison between Pork_Pea and pork groups by heatmap of discrepant metabolites (G); volcano plot of discrepant metabolites (H); discrepant metabolic signatures ranked by VIP score (I). The red spots or pieces indicate up-regulation, and the green ones indicate down-regulation.
Figure 10
Figure 10
Alterations of specific cecal metabolites and pathways (A–C) Relative expression of discrepant metabolites: L-arginine (a-1), N2-succinyl-L-ornithine (a-2), anserine (a-3), hercynine (a-4), oxypinnatanine (a-5), glutamylproline (a-6), glycocholate (b-1), taurine (b-2), 3β,7α-dihydroxy-5-cholestenoate (b-3), 27-hydroxycholesterol (b-4), sphinganine (c-1), 17-hydroxylinolenic acid (c-2), 9,10-DiHODE (c-3), 12-HETE-GABA (c-4), 1α,25-dihydroxy-11alpha-[(1R)-oxiranyl]vitaminD3 (c-5). (D–F) The main metabolic pathways according to integrative analysis of the pathway impact and p value of metabolic signatures in comparison of pork and pea (D), Pea_Pork and pea (E), pork and Pork_Pea (F). (G) A diagram of major relevant pathway of arginine and histidine metabolism. Green color of metabolites represents the up-regulation and red color represents the down-regulation in the presence of pea protein. Arrow with different color represents significantly different pathway observed in comparison of different groups: orange represents pork versus pea, Pea_Pork versus pea, pork versus Pork_Pea; purple represents pork versus pea, Pea_Pork versus pea; blue represents pork versus pea, pork versus pork_Pea. (H) A diagram of major relevant pathway of primary bile acid biosynthesis. Green and red metabolites represent up-regulation and down-regulation in the presence of pea protein. Arrow with different color represents significantly different pathways observed in comparison of different groups: purple represents pork versus pea, Pea_Pork versus pea; yellow represents Pea_Pork versus pea, respectively. (I) Correlation analysis of specific metabolites with gut microbiota at family level. (J) Correlation analysis of specific metabolites with serum lipid profiles. Statistical significance was calculated by Student's t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant). All error bars indicate SE.
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